The present invention relates to a device and method for measuring the level of hemoglobin in a blood sample. The device comprises a disposable electrochemical cell containing an agent which lyses red blood cells and a reagent capable of being reduced by hemoglobin.
Hemoglobin is a respiratory molecule found in red blood cells. It is responsible for transporting oxygen from the lungs to body cells and for transporting carbon dioxide from body cells to the lungs. Hemoglobin has a molecular weight of 68,000 and contains four polypeptide chains. Each chain binds to a heme group which consists of a tetrapyrrole ring chelated to an Fe+2 ion. In the lungs, the iron atoms of the hemoglobin molecule reversibly combine with an oxygen molecule, which is then transported to body cells as blood circulates. The oxygen is released from the hemoglobin molecule in the tissues, then the oxygen-free hemoglobin molecule picks up carbon dioxide which is transported back to the lungs, where it is released.
Hemoglobin is produced in cells in the bone marrow that become red blood cells. Certain illnesses result in a deficiency of hemoglobin, such as anemia and sickle cell disease. Still other diseases, such as polycythemia or erythrocytosis, result in excessive levels of hemoglobin. Therefore, as an aid in the diagnosis or monitoring of such diseases, a method and device for determining the concentration of hemoglobin in whole blood is desirable.
Numerous methods and devices for the determination of hemoglobin are known. These methods include both direct analysis, i.e., analysis without prior modification of the hemoglobin, and indirect analysis. An example of a direct analysis method is the Tallquist Method, wherein a measurement of the transmission or reflection optical density of the red color imparted by oxyhemoglobin, one form of hemoglobin, is obtained. An example of an indirect analysis method is Drabkin""s Method. In this method, the iron in hemoglobin is oxidized with a ferricyanide to form methemoglobin, which is converted with a cyanide to cyanmethemoglobin, which is then measured spectrometrically. Both of these methods have the disadvantage of requiring expensive analytical instrumentation and complicated sample preparation. Therefore, a quick, simple, and inexpensive device and method for measuring hemoglobin that overcomes the deficiencies of prior art methods is desirable.
The present invention provides a device and method for measuring hemoglobin with a disposable sensing element, suitable for a single use, that can be combined with a meter to give a robust, fast, and easy to use test that is amenable to field as well as laboratory use. In particular, the invention relates to the use of an electrochemical sensor that utilizes a redox agent that reacts with hemoglobin to produce an electrochemically detectable signal. The method of the present invention measures total hemoglobin, oxygenated plus unoxygenated, in contrast to spectrophotometric methods wherein the hemoglobin must be converted to a single form in a separate chemical step, e.g., oxidation of hemoglobin containing Fe+2 to methemoglobin containing Fe+3. Measurement of hemoglobin by the method of the present invention is not dependent upon the extent of glycosylation or oxygenation of the hemoglobin present in the sample.
In a first aspect of the present invention, a device for detecting a presence or an absence of hemoglobin in an aqueous sample is provided, the device including an electrochemical cell having a sensing chamber, a first electrode, a second electrode, an aperture for admitting the sample into the sensing chamber, and a reagent contained within the sensing chamber wherein the reagent is capable of being reduced by hemoglobin to generate an electrical signal indicative of the presence or absence of hemoglobin. The electrochemical cell may be designed to be disposed of after use in a single experiment.
In one aspect of this embodiment, first electrode is a sensing electrode. The sensing electrode may be platinum, palladium, carbon, indium oxide, tin oxide, gold, iridium, copper, steel, silver, or mixtures thereof The first electrode may be formed by a technique including sputtering, vapor coating, screen printing, thermal evaporation, ink jet printing, ultrasonic spraying, slot coating, gravure printing or lithography.
In another aspect of this embodiment, the second electrode is a counter electrode. The second electrode may be a metal in contact with a metal salt, for example, silver in contact with silver chloride, silver in contact with silver bromide, silver in contact with silver iodide, mercury in contact with mercurous chloride, and mercury in contact with mercurous sulfate. The second electrode may also be a reference electrode.
In another aspect of this embodiment, the electrochemical cell further includes a third electrode, which may be a reference electrode. The third electrode may include a metal in contact with a metal salt, for example, silver in contact with silver chloride, silver in contact with silver bromide, silver in contact with silver iodide, mercury in contact with mercurous chloride, and mercury in contact with mercurous sulfate.
In another aspect of this embodiment, the reagent may include dichromate, vanadium oxides, permanganate, electroactive organometallic complexes, quinones, dichlorophenolindophenol, and ferricyanide. A buffer, such as a phosphate, carbonate, alkali metal salt of mellitic acid, or alkali metal salt of citric acid, may be contained within the sensing chamber. The sensing chamber further includes a red blood cell lysing agent, for example, one selected from ionic detergents, nonionic detergents, proteolytic enzymes, lipases, saponin, sodium dodecyl sulfate, cetyl trimethylammonium bromide, or polyethoxylated octylphenol.
In another aspect of this embodiment, the sample includes whole blood.
In another aspect of this embodiment, the sensing chamber further includes a support contained within the sensing chamber, for example, mesh, nonwoven sheet, fibrous filler, macroporous membrane, sintered powder, or combinations thereof. The reagent, red blood cell lysing agent, and/or buffer may be contained within or supported on the support.
In another aspect of this embodiment, the second electrode is mounted in opposing relationship a distance of less than about 500 microns from the first electrode; less than about 150 microns from the first electrode; or less than about 150 microns and greater than about 50 microns from the first electrode.
In another aspect of this embodiment, the device includes an interface for communication with a meter. The interface may communicate a voltage or a current. The electrochemical cell may be a thin layer electrochemical cell.
In a second aspect of the present invention, a method for detecting a presence or an absence of hemoglobin in an aqueous sample is provided, the method including providing a device including an electrochemical cell having a sensing chamber, a first electrode, a second electrode, an aperture for admitting the sample into the sensing chamber, and a reagent contained within the sensing chamber, wherein the reagent is capable of being reduced by hemoglobin to generate an electrical signal indicative of the presence or absence of hemoglobin; providing an aqueous sample; allowing the sample to flow through the aperture and into the sensing chamber, such that the first and second electrodes are substantially covered; and obtaining an electrochemical measurement indicative of the presence or absence of hemoglobin present in the sample.
In one aspect of this embodiment, the electrochemical cell is designed to be disposed of after use in a single experiment, or may be a thin layer electrochemical cell. The electrochemical measurement may be an amperometric measurement, a potentiometric measurement, a coulometric measurement, or a quantitative measurement.
In a third aspect of the present invention, a method is provided for measuring hemoglobin in a fluid whole blood sample, the whole blood sample containing red blood cells, the red blood cells containing hemoglobin, wherein the method includes providing a device including an electrochemical cell having a sensing chamber, a first electrode, a second electrode, an aperture for admitting the sample into the sensing chamber; a reagent contained within the sensing chamber and capable of being reduced by hemoglobin; and a red blood cell lysing agent contained within the sensing chamber; placing the whole blood sample in the sensing chamber, whereby the lysing agent contained within the sensing chamber releases hemoglobin from the red blood cells, whereby the hemoglobin thus released reduces the reagent; and obtaining an electrochemical measurement indicative of the level of hemoglobin present in the whole blood sample.
In one aspect of this embodiment, the electrochemical cell is designed to be disposed of after use in a single experiment, or may be a thin layer electrochemical cell.
In one aspect of this embodiment, the method further includes obtaining an electrochemical measurement indicative of the presence or absence of hemoglobin in the sample by applying a negative potential to the first electrode; measuring a current generated by reaction of the reagent and hemoglobin; analyzing the current to give a result, the result including a time required for substantial lysis of the red blood cells or a derived final value for the current; calculating a percentage of the reaction completed as a function of time based on the result of the analyzing step; reversing the potential on the first electrode; measuring a transient current; and determining a diffusion coefficient and a concentration of a reduced form of the reagent based on the transient current.
In a fourth aspect of the present invention, a method of manufacture of a device for detecting the presence or absence of hemoglobin in an aqueous sample is provided, the device including an electrochemical cell having a sensing chamber, a first electrode, a second electrode, an aperture for admitting the sample into the sensing chamber, and a reagent contained within the sensing chamber, and wherein the reagent is capable of being reduced by hemoglobin to generate an electrical signal indicative of the presence or absence of hemoglobin, the method including forming an aperture extending through a sheet of electrically resistive material, the aperture defining a side wall of the sensing chamber; mounting a first layer having a first electrode to a first side of the sheet and extending over the aperture whereby to define a first sensing chamber end wall, the first electrode facing the first side of the sheet; mounting a second layer having a second electrode to a second side of the sheet and extending over the aperture whereby to define a second sensing chamber end wall in substantial overlying registration with the first layer, the second electrode facing the second side of the sheet, whereby the sheet and layers form a strip; providing an aperture in the strip to permit entry of sample into the sensing chamber; and providing a reagent capable of being reduced by hemoglobin, wherein the reagent is contained within the sensing chamber.
In one aspect of this embodiment, the method further includes the step of providing a vent in the strip to permit the escape of air displaced from the sensing chamber as sample fills the sensing chamber.
In another aspect of this embodiment, the aperture is of a rectangular cross-section.
In another aspect of this embodiment, at least one of the electrodes includes a noble metal, such as palladium, platinum, and silver. At least one of the electrodes may be a sputter coated metal deposit. The electrodes may be adhered to the sheet, for example, by an adhesive such as a heat activated adhesive, pressure sensitive adhesive, heat cured adhesive, chemically cured adhesive, hot melt adhesive, and hot flow adhesive.
In another aspect of this embodiment, the method may include the step of providing a buffer and/or a red blood cell lysing agent contained within the sensing chamber. The reagent and/or buffer may be printed onto at least one wall of the sensing chamber. A support contained within the sensing chamber may also be provided, such as mesh, fibrous filler, macroporous membrane, sintered powder, or combinations thereof. The reagent may be supported on or contained within the support.
In another aspect of this embodiment, at least the sheet or one of the layers includes a polymeric material selected from polyester, polystyrene, polycarbonate, polyolefin, and mixtures thereof, or polyethylene terephthalate.
In another aspect of this embodiment, the second electrode is mounted in opposing relationship a distance of less than about 500 microns from the first electrode; less than about 150 microns from the first electrode; or less than about 150 microns and greater than about 50 microns from the first electrode.
In another aspect of this embodiment, the electrochemical cell is designed to be disposed of after use in a single experiment.